The invention relates generally to the field of organ and tissue transplantation, and more specifically to procedures for matching donor tissues and recipients whose bodies will not reject the tissue if transplanted into the recipient.
Transplantation of organs and tissues from the body of a donor into the body of a recipient has become a relatively common procedure. Owing to fundamental functions of the immune system of vertebrates (i.e., to alert the body to “non-self” materials and to assist in elimination of such non-self materials), tissues cannot be simply transplanted from the body of one individual of a given species into the body of any other individual of the same species without the possibility of immune complications. If the transplanted tissue is not compatible with the immune system of the recipient, the recipient's immune system can mount a response against the transplanted tissue whereby the transplanted tissue is destroyed. In order to avoid such transplant rejection, two basic strategies can be employed. First, one or more immune functions of the recipient can be inhibited, so as to lessen or eliminate the immune response mounted against the transplant. Second, the transplant and recipient can be screened and matched, to lessen the likelihood and/or severity of a recipient immune response against the transplant. The subject matter described herein is directed primarily toward this second strategy, although the two strategies are not mutually exclusive—both can be employed in the same transplantation.
It has long been known that cells in vertebrate tissues express antigens on their surface, and that these antigens can vary significantly among individuals of the same species. For example, human cells display proteins designated human leukocyte antigens (HLAs) on their surfaces. For any individual human, the HLAs displayed by that individual's cells are recognized as “self” by the individual's immune system, and the individual's immune system does not (normally) mount an immune response against his or her own cells. However, the HLAs displayed by different individual humans can vary significantly enough that HLAs not normally expressed by an individual's cells, such as HLAs of transplanted tissue, can be recognized as “non-self” by an individual's immune system, leading to initiation of an immune response directed against the transplanted tissue. If the immune response initiated against a transplanted tissue is sufficiently severe to induce death of most or all transplanted cells or destruction of sufficient transplanted extracellular material, the transplanted tissue can fail to exhibit desirable functions or properties that were the reason for the transplant. That is, the goals which led to the transplantation can fail to be achieved if the recipient immune response mounted against the transplanted tissue is sufficiently severe.
One type of immune response that a recipient's body can mount against a transplanted tissue depends on binding of antibodies produced by the recipient's body to antigens (e.g., HLAs) that are displayed on surfaces of the transplanted tissue. Specific binding between antibodies and their corresponding antigens can catalyze a reaction (“complement fixation”) that leads to induction of a potent cytotoxic immune response directed against the transplanted tissue. Analyzing the ability of recipient immune system components to detect donor tissue as “non-self” and mount a cytotoxic response and selecting donor-recipient pairs to avoid such reactions is referred to generally as “crossmatching” and is discussed extensively in the literature (see, e.g., Mulley et al., 2011, Nephrology 16:125-133).
One known crossmatching technique involves contacting lymphocytes obtained from the donor of a potential transplant tissue with blood serum obtained from a proposed recipient of the transplant tissue. Serum includes antibodies which circulate in the blood of the recipient. If the recipient's serum includes antibodies which bind specifically with antigens which appear on donor lymphocytes, such binding can be detected. In one common detection technique known as “flow crossmatching,” donor lymphocytes are contacted with recipient serum for a period of time, after which non-bound antibodies are removed by separating the lymphocytes and serum and rinsing the lymphocytes with an excess reagent. The lymphoctyes are then contacted with a labeled reagent capable of detecting recipient antibodies (e.g., a fluoresceinated antibody which binds to human antibodies, optionally of a particular type, such as IgM or IgG, or subtype, such as IgG1, IgG2, IgG3, or IgG4) and thereafter rinsing non-bound labeled reagent from the lymphocytes. Binding of recipient antibodies with donor lymphocytes is detected by suspending the lymphocytes in a fluid and passing them through a flow cytometer capable of detecting the label of the labeled reagent. Detection of the label together with a lymphocyte by the flow cytometer (e.g., detection of fluorescence corresponding to fluorescein) indicates that one or more recipient antibodies recognized by the labeled reagent is bound to the lymphocyte. Such bound recipient antibodies are commonly designated “anti-donor antibodies” (ADAbs). If multiple flow crossmatching reactions are performed, each using a labeled reagent having specificity for a different recipient antibody type and/or subtype, a profile of the type(s) and subtypes(s) of ADAbs that are present in an potential recipient's serum can be developed.
It has been observed that ADAb types and subtypes can influence the likelihood that a transplanted tissue will be rejected by a recipient. Mulley et al., for example, recognized that IgG4 does not activate complement and that potential recipients whose ADAb detected using donor leukocytes are solely of the IgG4 subtype may be less likely to activate complement in vivo (i.e., indicating a potentially suitable donor-recipient match). Comparable observations were reported by Cicciarelli et al. (U.S. patent application publication no. 2010/0261203). Gao et al. (2014, Am. J. Transplant. 14(7):1581-1591) recognized that IgG antibodies of subtypes IgG1 and IgG3 which react with apoptotic cells are more likely to lead to late rejection of transplanted kidneys, presumed by those authors to be attributable to the complement-fixing ability of IgG1 and IgG3 subtypes. However, previous workers examined binding between antigen-coated beads and antibodies in recipient serum, which assays can be laborious and not necessarily indicative of interactions between recipient antibodies and donor cells.
Improved crossmatching assays capable of quickly and accurately determining the suitability of a potential tissue transplant for implantation in (or on) an individual recipient would be desirable. This disclosure describes such assays.